Project 1: Myosin activity facilitates Vinculin recruitment to focal adhesions through modulation of paxillin phosphorylation[unreadable] [unreadable] Personell: Ana-Maria Pasapera-Limon[unreadable] [unreadable] Focal Adehsion mediate cell migration by linking the contractile actomyosin cytoskeleton to the ECM. Upon cell binding to the ECM, integrin receptors recruit a complex of proteins leading to the formation of small focal complexes that grow in size and change in composition, in a loosely-defined process known as FA maturation. It is well established that myosin-II mediated contractile force in the cytoskeleton drives maturation. However, which proteins specifically respond to this mechanical signal via changes in their residence or post-tranlational state within FAs is poorly defined. To approach this question, we treated mouse embryonic fibroblasts (MEF) with inhibitors of myosin II-dependent contraction and used immunofluorescence microscopy, FRAP, immunoprecipitation and western blot analyses to examine changes in localization and phosphorylation status of key FA proteins. As reported, inhibition of contraction with either the ROCK inhibitor Y-27623 or the myosin-II ATPase inhibitor blebbistatin reduced FA size, promoted rapid cell migration with tail retraction defects, and shortened FA lifetime. Immunofluorescence analysis of myosin-II inhibited cells revealed no change in the level or paxillin, FAK or talin in adhesions, but loss of the proteins vinculin, a-actinin and zyxin, indicating their myosin-II dependent recruitment to FA. FRAP analysis of fluorescent protein conjugates of several FA proteins revealed the actin-binding protein vinculin as a very stable component of mature adhesions, therefore we focused on the mechanism of it myosin-II dependent recruitment to FA. Examination of vinculin and paxillin localization in cells spreading on ECM-coated glass or plated on soft (4kPA) ECM-coated polyacrylamide gels revealed colocalization only in a subset of FA, indicating that physiological reduction in myosin II contraction also can result in loss of vinculin from some FA. We found that vinculin and FAK exhibit myosin II- dependent association with paxillin immunoprecipitates. Src- and FAK-mediated phosphorylation of paxillin on Y31 and Y118 was also found to be myosin II-dependent. These results suggest that vinculin may be recruited to FA via association with phospho-paxillin. To test this, we overexpressed paxillin Y31E phospomimic mutant and Y31F non-phosphorylatable mutant and examined vinculin localization. This revealed vinculin localized at FA in myosin II-inhibited cells expressing paxillin Y31E, but not Y31F, indicating that the Y31E mutation can override the requirement for myosin II activity in vinculin recruitment to FA. Together, these results suggest that myosin-II activity promotes FAK/Src-mediated paxillin phosphorylation to drive the recruitment of vinculin to FA during tensionmedited FA maturation.[unreadable] [unreadable] This work has been presented as posters at the American Society for Cell Biology and the Fronteirs in Cell Migration meetings and is currently being prepared for publication[unreadable] [unreadable] Project 2: PROTEOMIC ANALYSIS OF FOCAL ADHESION MATURATION[unreadable] [unreadable] Personell: Jean-Cheng Kuo[unreadable] [unreadable] Focal adhesions (FA) are complex plasma-membrane associated macromolecular assemblies that serve to physically connect cells to, as well as transducer signals to and from, the surrounding extracellular matrix (ECM). FA play a crucial role in the control of tissue structure and morphogenesis as well as cell motility. It is well established that FA undergo a maturation process in which their size increases that is regulated by either small GTPase signaling or by force applied to the FA. Indeed, Rac1 signalling or reduced force promotes formation of small FA, while RhoA signalling or high force promotes FA growth and maturation. The maturation of FA is thought to be critical to modulating the signals transduced from the ECM into the cell that regulate cell growth and differentiation. However, how FA maturation achieves signal modulation is not known. Here, we test the hypothesis that FA maturation alters FA protein composition, and further, that physical and biochemical pathways to FA maturation may not necessarily be the same. We developed a systematic method for biochemical isolation of focal adhesions from human fibroblasts, followed by analysis of the FA protein compositions by the Mud-pit LC-MS proteomics method. To test our hypothesis, we modulated FA maturation in several ways and analyzed FA protein composition. To alter the force on FA, we either treated cells with the myosin II inhibitor blebbistatin or cultured cells on polyacrylamide sustrates of different stiffnesses. To alter FA maturation biochemically, we expressed constitutively active mutants of RhoA (V14) or Rac1 (V12). This study provides a global view how FA composition is altered by physical and biochemical cues, and provides insight into how physical signals alter focal adhesion-related signaling networks to trigger diverse physiological functions. [unreadable] [unreadable] This work was performed in collaboration with John Yates and Xuemei Han (Scripps) and was presented at meetings of the American Society for Cell Biology, Gordon conference on Signalling from Adhesion Receptors, and Frontiers in Cell Migration meeting. It is ongoing.[unreadable] [unreadable] Project 3: Analysis of traction stress variation across single focal adhesions.[unreadable] [unreadable] Personnell: Sergey Plotnikov[unreadable] The ability of eukaryotic cells to generate force and sense the mechanical properties of the extracellular matrix (ECM) underlies many biological processes, such as cell migration, proliferation and differentiation. This is achieved in part by integrin-mediated focal adhesions (FA), protein assemblies that couple contractile actomyosin bundles to the ECM and transmit force generated by the cytoskeleton across the plasma membrane. It has been recently demonstrated that protein composition and/or post-translational modification state can vary across an individual FA. However, whether this translates to variation in physiological properties and/or function for sub-FA domains is not known. We hypothesize that variation in biochemical composition across an individual FA mediates differences in force transmission across a FA that may be critical to it role in cell migration. To test this, we used high-resolution traction force microscopy (Sabass et al., 2008) to analyze the distribution of traction stresses along single FA and correlate this with the density of specific FA proteins. We identified two distinctive patterns of traction stress: for the majority of FAs, the region of maximum stress was located to the geometrical center of the FA, while for some FA it was significantly skewed toward their distal edges (cell periphery). Translocation of the traction force maximum from the FA center towards its distal edge usually occurred shortly before cell edge retraction. Analysis of GFP-paxillin intensity in FA revealed that maximum traction stress colocalized with maximum paxillin concentration, independent of the position of maximal stress within the FA. Next, we tested the hypothesis that mechanical stress applied on FA can induce translocation of the traction stress maxima. We found that centripetal pulling on a fibronectin-coated magnetic bead attached to the dorsal side of the cell caused displacement of traction force in ventral FA similar to that observed in FA in a retracting cell edge. These results suggest that FA biochemical and mechanical properties are directly coupled.[unreadable] [unreadable] This work was performed in colabboration with Benedikt Sabass and Ulrich Schwartz (university of Heidelberg) at the Marine Biological Laboratory in Woods HOle MA. It was presaented at the American Society for Cell Biology, Gordon conference on Signalling from Adhesion Receptors, and Frontiers in Cell